Structural and dynamic properties of the homodimeric hemoglobin from Scapharca inaequivalvis Thr-72-->Ile mutant: molecular dynamics simulation, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy studies.

Molecular dynamics simulations, low temperature visible absorption spectroscopy, and resonance Raman spectroscopy have been performed on a mutant of the Scapharca inaequivalvis homodimeric hemoglobin, where residue threonine 72, at the subunit interface, has been substituted by isoleucine. Molecular dynamics simulation indicates that in the Thr-72-->Ile mutant several residues that have been shown to play a role in ligand binding fluctuate around orientations and distances similar to those observed in the x-ray structure of the CO derivative of the native hemoglobin, although the overall structure remains in the T state. Visible absorption spectroscopy data indicate that in the deoxy form the Soret band is less asymmetric in the mutant than in the native protein, suggesting a more planar heme structure; moreover, these data suggest a similar heme-solvent interaction in both the liganded and unliganded states of the mutant protein, at variance with that observed in the native protein. The "conformation sensitive" band III of the deoxy mutant protein is shifted to lower energy by >100 cm-1 with respect to the native one, about one-half of that observed in the low temperature photoproducts of both proteins, indicating a less polar or more hydrophobic heme environment. Resonance Raman spectroscopy data show a slight shift of the iron-proximal histidine stretching mode of the deoxy mutant toward lower frequency with respect to the native protein, which can be interpreted in terms of either a change in packing of the phenyl ring of Phe-97, as also observed from the simulation, or a loss of water in the heme pocket. In line with this latter interpretation, the number of water molecules that dynamically enters the intersubunit interface, as calculated by the molecular dynamics simulation, is lower in the mutant than in the native protein. The 10-ns photoproduct for the carbonmonoxy mutant derivative has a higher iron-proximal histidine stretching frequency than does the native protein. This suggests a subnanosecond relaxation that is slowed in the mutant, consistent with a stabilization of the R structure. Taken together, the molecular dynamics and the spectroscopic data indicate that the higher oxygen affinity displayed by the Thr-72-->Ile mutant is mainly due to a local perturbation in the dimer interface that propagates to the heme region, perturbing the polarity of the heme environment and propionate interactions. These changes are consistent with a destabilization of the T state and a stabilization of the R state in the mutant relative to the native protein.     

Resilience of the iron environment in heme proteins

Conformational flexibility is essential to the functional behavior of proteins. We use an effective force constant introduced by Zaccai, the resilience, to quantify this flexibility. Site-selective experimental and computational methods allow us to determine the resilience of heme protein active sites. The vibrational density of states of the heme Fe determined using nuclear resonance vibrational spectroscopy provides a direct experimental measure of the resilience of the Fe environment, which we compare quantitatively with values derived from the temperature dependence of atomic mean-squared displacements in molecular dynamics simulations. Vibrational normal modes in the THz frequency range dominate the resilience. Both experimental and computational methods find a higher resilience for cytochrome c than for myoglobin, which we attribute to the increased number of covalent links to the peptide in the former protein. For myoglobin, the resilience of the iron environment is larger than the average resilience previously determined for hydrogen sites using neutron scattering. Experimental results suggest a slightly reduced resilience for cytochrome c upon oxidation, although the change is smaller than reported in previous Mössbauer investigations on a bacterial cytochrome c, and is not reproduced by the simulations. Oxidation state also has no significant influence on the compressibility calculated for cyt c, although a slightly larger compressibility is predicted for myoglobin.     

Molecular dynamics simulations of Hydrogenobacter thermophilus cytochrome c552: comparisons of the wild-type protein, a b-type variant, and the apo state

Molecular dynamic simulations have been performed for wild-type Hydrogenobacter thermophilus cytochrome c(552), a b-type variant of the protein, and the apo state with the heme prosthetic group removed. In the b-type variant, Cys 10 and Cys 13 were mutated to alanine residues, and so the heme group was no longer covalently bound to the protein. Two 8-ns simulations have been performed for each system at 298 and 360 K. The simulations of the wild-type protein at 298 K show a very close agreement with experimental NMR data. A fluxional process involving the side chain of Met 59, which coordinates to the heme iron, is observed in accord with proposals from NMR studies. Overall, the structure and dynamical behavior of the protein during the simulations of the b-type variant is closely similar to that of the wild-type protein. However, side chains in the heme-binding site show larger fluctuations in the b-type variant simulation at 360 K. In addition, structural changes are seen for a number of residues close to the heme group, particularly Gly 22 and Ser 51. The simulations of the apo state show significant conformational changes for residues 50-59. These residues form a loop region, which packs over the heme group in the wild-type protein and hydrogen bonds to the heme propionate groups. In the absence of heme, in the apo state simulations, these residues form short but persistent regions of beta-sheet secondary structure. These could provide nucleation sites for the conversion to amyloid fibrils.     

Redox thermodynamics of the Fe(III)/Fe(II) couple of human myeloperoxidase in its high-spin and low-spin forms

Myeloperoxidase (MPO) (donor, hydrogen peroxide oxidoreductase, EC 1.11.1.7) is the most abundant neutrophil enzyme and catalyzes predominantly the two-electron oxidation of ubiquitous chloride (Cl-), to generate the potent bleaching oxidant hypochlorous acid (HOCl), thus contributing to bacterial killing and inflammatory reactions of neutrophils. Here, the thermodynamics of the one-electron reduction of the ferric heme in its ferric high-spin and cyanide-bound low-spin forms were determined through spectroelectrochemical experiments. The E(o)' values for free and cyanide-bound MPO (5 and -37 mV, respectively, at 25 degrees C and pH 7.0) are significantly higher than those of other heme peroxidases. Variable-temperature experiments revealed that the enthalpic stabilization of ferric high-spin MPO is much weaker than in other heme peroxidases and is exactly compensated by the entropic change upon reduction. In contrast to those of other heme peroxidases, the stabilization of the ferric cyanide-bound MPO is also very weak and fully entropic. This peculiar behavior is discussed with respect to the MPO-typical covalent heme to protein linkages as well as to the published structures of ferric MPO and its cyanide complex and the recently published structure of lactoperoxidase as well as the physiological role of MPO in bacterial killing.     

Effect of prototypic drugs ibuprofen and warfarin on global chaotropic unfolding of human serum heme-albumin: a fast-field-cycling 1H-NMR relaxometric study

Human serum albumin (HSA) is the most prominent protein in plasma, but it is also found in tissues and secretions throughout the body. The three-domain design of HSA provides a variety of binding sites for many ligands, including heme and drugs. HSA has been used as a model multidomain protein to investigate how interdomain interactions affect the global folding/unfolding process. Here, we report on the reversible chemical denaturation of heme-HSA involving three different conformational states (F, N, and B, occurring at pH 4.0, 7.0, and 9.0, respectively) and on the effect of prototypic drugs ibuprofen and warfarin on thermodynamics of the reversible unfolding process. Chaotropic unfolding of heme-HSA in the F, N, and B conformations is governed by different thermodynamic regimes, with the B form showing an entropic stabilization of the structure that compensates an enthalpic destabilization, and the F form easily unfolding under entropic control. Warfarin and ibuprofen binding stabilizes heme-HSA in both N and B states.     

The effect of heme on the conformational stability of micro-myoglobin

Micro-myoglobin, the isolated heme-binding subdomain of myoglobin, is a valuable model system for the investigation of heme recognition and binding by proteins, and provides an example of protein folding induced by cofactor binding. Theoretical studies by molecular dynamics simulations on apo- and holo-micro-myoglobin show that, by contrast with the case of the full-length wild-type protein and in agreement with earlier experimental evidence, the apo-protein is not stably folded in a native-like conformation. With the cofactor bound, however, the protein fragment maintains its folded conformation over 1.5 ns in molecular dynamics simulations. Further inspection of the model structures reveals that the role of heme in stabilizing the folded state is not only a result of its direct interactions with binding residues (His93, Arg45 and Lys96), but also derives from its shielding effect on a long-range electrostatic interaction between Arg45 and Asp60, which, in the molecular dynamics simulations, apparently triggers the unfolding process of apo-micro-myoglobin.     

Characterization of the structure-activity relationships of rat types I and II 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase by site-directed mutagenesis and expression in HeLa cells

The membrane-bound enzyme 3 beta-hydroxysteroid dehydrogenase/delta 5 -delta 4 isomerase (3 beta-HSD) catalyzes the conversion of delta 5 -3 beta-hydroxysteroid precursors into delta 4-ketosteroids, thus representing an essential step in the biosynthesis of all classes of hormonal steroids. We have recently characterized two types of cDNA clones encoding rat 3 beta-HSD proteins, the rat type I protein being much more active than type II. In order to characterize further the functional difference between these two 3 beta-HSD types, transient expression of type I and type II 3 beta-HSD cDNAs was performed in HeLa human cervical carcinoma cells. The present study demonstrates that the type I 3 beta-HSD protein has a relative specificity 64- and 46-fold higher than type II protein for pregnenolone (PREG) and dehydroepiandrosterone (DHEA) as substrates, respectively. The Km values of type I and type II enzymes were calculated at 0.74 and 14.3 microM, respectively, using PREG as substrate whereas the respective Km values were 0.68 and 12.9 microM when DHEA was used, thus showing that their different relative specificity results largely from a different affinity for substrates. Since the change of 4 amino acid residues in type II could prevent the formation of a putative membrane-spanning domain (MSD) predicted between amino acid residues 75 and 91, chimeric cDNAs containing either type I MSD in type II (II + MSD) or an absence of this MSD in type I (I-MSD) were constructed and transiently expressed. The addition of MSD intype II 3 beta-HSD markedly increased the affinity leading to Km values similar to those found in type I 3 beta-HSD, namely 0.36 and 0.40 microM for PREG and DHEA, respectively. II + MSD chimera thus encodes a protein having a relative specificity for PREG and DHEA of 58 and 73%, respectively, to that of native type I 3 beta-HSD. Moreover, removal of MSD in the type I protein (I-MSD chimera) decreased the relative specificity of type I 3 beta-HSD protein for PREG and DHEA to only 0.37 and 0.48%, with respective Km values of 11.7 and 11.0 microM, thus strongly indicating the functional importance of this putative MSD which is predicted in wild type rat type I as well as in macaque and human 3 beta-HSD proteins.     

Spectroscopic characterization of heme A reconstituted myoglobin.

The focus of this study was to examine the functional role of the unusual peripheral substitution of heme A. The effects of heme A stereochemistry on the reconstitution of the porphyrin have been examined in the heme A-apo-myoglobin complex using optical absorption and resonance Raman and electron paramagnetic resonance spectroscopies. The addition of one equivalent of heme A to apo-Mb produces a complex which displays spectroscopic signals consistent with a distribution of high- and low-spin heme chromophores. These results indicate that the incorporation of heme A into apo-Mb significantly perturbs the protein refolding.     

Dependence of Protein Crystal Stability on Residue Charge States and Ion Content of Crystal Solvent

Protein crystallization is frequently induced by the addition of various precipitants, which directly affect protein solubility. In addition to organic cosolvents and long-chain polymers, salts belong to the most widely used precipitants in protein crystallography. However, despite such widespread usage, their mode of action at the atomistic level is still largely unknown. Here, we perform extensive molecular dynamics simulations of the villin headpiece crystal unit cell to examine its stability at different concentrations of sodium sulfate. We show that the inclusion of ions in crystal solvent at high concentration can prevent large rearrangements of the asymmetric units and a loss of symmetry of the unit cell without significantly affecting protein dynamics. Of importance, a similar result can be achieved by neutralizing several specific charged residues suggesting that they may play an active role in crystal destabilization due to unfavorable electrostatic interactions. Our results provide a microscopic picture behind salt-induced stabilization of a protein crystal and further suggest that adequate modeling of realistic crystallization conditions may be necessary for successful molecular dynamics simulations of protein crystals.     

Free energy of transition for the individual alkaline conformers of yeast iso-1-cytochrome c

Direct protein electrochemistry was used to obtain the thermodynamic parameters of transition from the native (state III) to the alkaline (state IV) conformer for untrimethylated Saccharomyces cerevisiae iso-1-cytochrome c expressed in E. coli and its single and multiple lysine-depleted variants. In these variants, one or more of the lysine residues involved in axial Met substitution (Lys72, Lys73, and Lys79) was mutated to alanine. The aim of this work is to determine the thermodynamic affinity of each of the substituting lysines for the heme iron and evaluate the interplay of enthalpic and entropic factors. The equilibrium constants for the deprotonation reaction of Lys72, 73, and 79 were computed for the minimized MD average structures of the wild-type and mutated proteins, applying a modified Tanford-Kirkwood calculation. Solvent accessibility calculations for the substituting lysines in all variants were also performed. The transition enthalpy and entropy values within the protein series show a compensatory behavior, typical of a process involving extensive solvent reorganization effects. The experimental and theoretical data indicate that Lys72 most readily deprotonates and replaces M80 as the axial heme iron ligand, whereas Lys73 and Lys79 show comparably higher pKa values and larger transition free energies. A good correlation is found within the series between the lowest calculated Lys pKa value and the corresponding experimental pKa value, which can be interpreted as indicative of the deprotonating lysine itself acting as the triggering group for the conformational transition. The triple Lys to Ala mutant, in which no lysine residues are available for heme iron binding, features transition thermodynamics consistent with a hydroxide ion replacing the axial methionine ligand.     

Coarse-grained simulations of conformational dynamics of proteins: Application to apomyoglobin

A coarse-grained dynamic Monte Carlo method is proposed for investigating the conformational dynamics of proteins. Each residue is represented by two interaction sites, one at the α-carbon, and the other on the amino acid sidechain. Geometry and energy parameters extracted from databank structures are used. The method is applied to the crystal structure of apomyoglobin (apo-Mb). Equilibrium and dynamic properties of apo-Mb are characterized within computation times one order of magnitude shorter than conventional molecular dynamics (MD) simulations. The calculated rms fluctuations in α-carbons are in good agreement with crystallographic temperature factors. Regions exhibiting enhanced conformational mobilities are identified. Among the loops connecting the eight helices A to H, the loop CD undergoes the fastest motions, leading to partial unwinding of helix D. Helix G is the most stable helix on the basis of the kinetic stability of dihedral angles, followed by the respective helices A, E, H, and B. These results, in agreement with H/D exchange and two-dimensional NMR experiments, as well as with MD simulations, lend support to the use of the proposed approach as an efficient, yet physically plausible, means of characterizing protein conformational dynamics. Proteins 31:271–281, 1998. © 1998 Wiley-Liss, Inc.     

Entropic stabilization of proteins and its proteomic consequences

Evolutionary traces of thermophilic adaptation are manifest, on the whole-genome level, in compositional biases toward certain types of amino acids. However, it is sometimes difficult to discern their causes without a clear understanding of underlying physical mechanisms of thermal stabilization of proteins. For example, it is well-known that hyperthermophiles feature a greater proportion of charged residues, but, surprisingly, the excess of positively charged residues is almost entirely due to lysines but not arginines in the majority of hyperthermophilic genomes. All-atom simulations show that lysines have a much greater number of accessible rotamers than arginines of similar degree of burial in folded states of proteins. This finding suggests that lysines would preferentially entropically stabilize the native state. Indeed, we show in computational experiments that arginine-to-lysine amino acid substitutions result in noticeable stabilization of proteins. We then hypothesize that if evolution uses this physical mechanism as a complement to electrostatic stabilization in its strategies of thermophilic adaptation, then hyperthermostable organisms would have much greater content of lysines in their proteomes than comparably sized and similarly charged arginines. Consistent with that, high-throughput comparative analysis of complete proteomes shows extremely strong bias toward arginine-to-lysine replacement in hyperthermophilic organisms and overall much greater content of lysines than arginines in hyperthermophiles. This finding cannot be explained by genomic GC compositional biases or by the universal trend of amino acid gain and loss in protein evolution. We discovered here a novel entropic mechanism of protein thermostability due to residual dynamics of rotamer isomerization in native state and demonstrated its immediate proteomic implications. Our study provides an example of how analysis of a fundamental physical mechanism of thermostability helps to resolve a puzzle in comparative genomics as to why amino acid compositions of hyperthermophilic proteomes are significantly biased toward lysines but not similarly charged arginines.     

Molecular dynamics simulations to determine the optimal loop length in the helix-loop-helix motif

In this study, 100 ps molecular dynamics simulations were conducted to determine the optimal length of the Gly loop in the helix-loop-helix motif. The results revealed that the length of the loop significantly affected the stability of the two alpha-helices. In addition, the optimal loop length to maintain the highest helicity of the target peptide was found to be the one corresponding to 7 Gly residues. Longer loop length resulted in structural destabilization due to the entropic effect, while shorter loop length was not suitable to achieve intrachain packing of the two alpha-helices.     

Key interactions in neocarzinostatin,a protein of the immunoglobulin fold family

Neocarzinostatin (NCS) is a seven-stranded beta-sandwich protein, the folding of which is similar to that of the variable domains of immunoglobulins (Ig). The investigation of the backbone dynamics of apo-NCS [Izadi-Pruneyre et al. (2001) Protein Sci., 10, 2228-2240] enabled us to identify the involvement of long side-chain residues in maintaining the rigidity of this beta-protein. In the perspective of using this protein for drug targeting, this raises the following question: do these residues also play a key role in the stabilization of the beta-sheet? To investigate this problem, various genetically engineered variants were constructed by mutating these residues to amino acids with shorter aliphatic side chains. These substitutions have no effects on the global fold. However, an important destabilization of the protein, higher than that expected for a simple 'large-to-small' substitution of buried hydrophobic residues, is observed for three mutants, V34A, V21A and V95A. Interestingly, the nature of the residues in these positions is highly conserved in the other Ig-like proteins. The absence of an evolutionary relationship between NCS and the other Ig-like proteins strongly suggests that this hydrophobic core is characteristic of the Ig-fold itself.     

Collagen stabilization at atomic level: crystal structure of designed (GlyProPro)10foldon

In a designed fusion protein the trimeric domain foldon from bacteriophage T4 fibritin was connected to the C terminus of the collagen model peptide (GlyProPro)(10) by a short Gly-Ser linker to facilitate formation of the three-stranded collagen triple helix. Crystal structure analysis at 2.6 A resolution revealed conformational changes within the interface of both domains compared with the structure of the isolated molecules. A striking feature is an angle of 62.5 degrees between the symmetry axis of the foldon trimer and the axis of the triple helix. The melting temperature of (GlyProPro)(10) in the designed fusion protein (GlyProPro)(10)foldon is higher than that of isolated (GlyProPro)(10,) which suggests an entropic stabilization compensating for the destabilization at the interface.     

Unraveling the molecular basis for ligand binding in truncated hemoglobins: The trHbO Bacillus subtilis case

Truncated hemoglobins (trHbs) are heme proteins present in bacteria, unicellular eukaryotes, and higher plants. Their tertiary structure consists in a 2‐over‐2 helical sandwich, which display typically an inner tunnel/cavity system for ligand migration and/or storage. The microorganism Bacillus subtilis contains a peculiar trHb, which does not show an evident tunnel/cavity system connecting the protein active site with the solvent, and exhibits anyway a very high oxygen association rate. Moreover, resonant Raman results of CO bound protein, showed that a complex hydrogen bond network exists in the distal cavity, making it difficult to assign unambiguously the residues involved in the stabilization of the bound ligand. To understand these experimental results with atomistic detail, we performed classical molecular dynamics simulations of the oxy, carboxy, and deoxy proteins. The free energy profiles for ligand migration suggest that there is a key residue, GlnE11, that presents an alternate conformation, in which a wide ligand migration tunnel is formed, consistently with the kinetic data. This tunnel is topologically related to the one found in group I trHbs. On the other hand, the results for the CO and O₂ bound protein show that GlnE11 is directly involved in the stabilization of the cordinated ligand, playing a similar role as TyrB10 and TrpG8 in other trHbs. Our results not only reconcile the structural data with the kinetic information, but also provide additional insight into the general behaviour of trHbs. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Effect of redox state on unfolding energetics of heme proteins.

Both the enthalpic and entropic contributions to unfolding of three heme proteins, cytochrome b(562), cytochrome c and myoglobin, are larger for the reduced than for the oxidized form. Thus, the higher thermodynamic stability of a reduced, as compared to an oxidized, heme protein is the net result of a large increase of favorable enthalpy and a small increase in unfavorable entropy. Upon comparing the unfolding energetics of the heme proteins to those of other single-domain proteins I find that protein length is the primary determinant of the thermodynamics.     

Allosteric changes in protein structure computed by a simple mechanical model: hemoglobin T<-->R2 transition

Information on protein dynamics has been usually inferred from spectroscopic studies of parts of the proteins, or indirectly from the comparison of the conformations assumed in the presence of different substrates or ligands. While molecular simulations also provide information on protein dynamics, they usually suffer from incomplete sampling of conformational space, and become prohibitively expensive when exploring the collective dynamics of large macromolecular structures. Here, we explore the dynamics of a well-studied allosteric protein, hemoglobin (Hb), to show that a simple mechanical model based on Gaussian fluctuations of residues can efficiently predict the transition between the tense (T, unliganded) and relaxed (R or R2, O(2) or CO-bound) forms of Hb. The passage from T into R2 is shown to be favored by the global mode of motion, which, in turn is driven by entropic effects. The major difference between the dynamics of the T and R2 forms is the loss of the hinge-bending role of alpha(1)-beta(2) (or alpha(2)-beta(1)) interfacial residues at alpha Phe36-His45 and beta Thr87-Asn102 in the R2 form, which implies a decreased cooperativity in the higher affinity (R2) form of Hb, consistent with many experimental studies. The involvement of the proximal histidine beta His92 in this hinge region suggests that the allosteric propagation of the local structural changes (induced upon O(2) binding) into global ones occur via hinge regions. This is the first demonstration that there is an intrinsic tendency of Hb to undergo T-->R2 transition, induced by purely elastic forces of entropic origin that are uniquely defined for the particular contact topology of the T form.     

Surface salt bridges contribute to the extreme thermal stability of an FN3-like domain from a thermophilic bacterium

This study uses differential scanning calorimetry, X-ray crystallography, and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5°C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25, and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22°C. Crystal structures of the wild type (WT) and a thermally destabilized (?Tm ?19.7°C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the WT were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins.